Method and apparatus for observing biological specimens using a scanning electron microscope

ABSTRACT

A biological specimen is prepared for electron scanning by plunging it into liquid nitrogen or liquid Freon. The frozen specimen together with its holder is transferred into the preevacuation chamber of a scanning electron microscope and treated according to the purpose of observation. When this is completed, the specimen is transferred to the specimen stage in the specimen chamber, the stage being maintained cold. The scanning electron beam is applied and the specimen image is observed.

United States Patent Hasegawa et al.

1451 Sept. 25, 1973 Inventors: Yoichi Hasegawa; Yuji Nagasawa,

both of Tokyo, Japan OTHER PUBLICATIONS Scanning Electron Microscopy of Labile Biological Material..., Echlin et al. Scanning Electron Microscopy Conference, IIT Research Inst. 4/1970 Cryofacture of Biological Material, Haggis, Scanning Electron Microscopy Conference, IIT Research Inst., Chicago, Ill. April, 1970 [73] Assignee: Nihon Denshi Kabushiki Kaisha, Tokyo, Japan Primary Examiner-James W. Lawrence [22] Flled' 1972 Assistant ExaminerC. E. Church [21] Appl. No.: 232,405 Attorney-John M. Webb [30] Foreign Application Priority Data [57] ABSTRACT Mar. 16. 1971 Japan ..46/l7578 Oct. 29. 1971 Japan ..46/100577 A blologlcal Speclmen 15 P p for electron Scan- Nov. 2. 1971 Japan ..46/87408 g y plung ng it int liqui n t ogen or liquid Freon.

The frozen specimen together with its holder is trans- [52] U.S. Cl. 250/49.5 B f red into the pre-evacuation chamber of a scanning [51 Int. Cl. I'IOlj 37/18 l tron mi ro cope and treated according to the pur- Of Search A, B pose of bservation. when this is r pleted the specimen is transferred to the specimen stage in the speci- References Cited men chamber, the stage being maintained cold. The UNITED STATES PATENTS scanning electron beam is applied and the specimen 3.483.373 12/1969 Asmus 250/495 B Image Observed- 3.374.349 3/1968 Macres... 250/495 PE 2.417.213 3/1947 Picard 250/495 B 6 Claims, 7 Drawmg Figures kn! J i I I PAIEmmsEPw 3:61.109

SHEETlUF 4 6 I ROTARY 6 283;," PUMP gimp 4 6 2 4 J 2 ROTARY f I PUMP E I LDIFFUSION PUMP LDIFFUSION PUMP X 20 1:? T nk L I 2i--- PATENTEBSEPZSIQYK SHEET 30F 4 1 METHOD AND APPARATUS FOR OBSERVING BIOLOGICAL SPECIMENS USING A SCANNING ELECTRON MICROSCOPE BACKGROUND As compared with a conventional transmission type electron microscope, a scanning electron microscope has the advantage of being able to produce a stereo image of the specimen configuration. In this case, however, since bulk specimens are used, the presence of water removed by sublimation in the microscope column results in damage to the specimen which adversely affects the natural three-dimensional configuration. This particularly applies to biological and chemical samples where the water content is high. In an effort to solve this problem, quench freezing of the specimen has recently been introduced. Quench freezing has a twofold advantage in that in addition to eliminating the possibility of damage due to sublimation, the specimen surface requires no coating in order to carry out observation. It is, however, necessary to prevent frost from forming on the surface of the frozen specimen or alternatively to effectively remove the frost from said surface.

Accordingly, it is an advantage of this invention to prevent the formation of frost on the specimen surface or to facilitate the removal of frost forming on the specimen surface. It is another advantage of this invention to prevent the formation of frost on the cross-section surface of the specimen where it is desired to carry out observation of the section. It is yet another advantage of this invention to prevent frost from forming on the interim specimen stage housed in the pre-evacuation chamber or on the inner wall of the pre-evacuation chamber during specimen exchange.

BRIEF DESCRIPTION Briefly, according to this invention, a pre-evacuation chamber is provided adjacent the electron optical column of the scanning electron microscope. The sample is quenched in liquid gas to be frozen. Thereafter, the sample is transferred to the pre-evacuation chamber and manipulated prior to placement in the specimen stage of the microscope to avoid the formation of frost or to remove the frost on the sample surface.

In accordance with a preferred embodiment of this invention, the above objects are made possible by placing certain devices and mechanisms, depending on the object in view, in the pre-evacuation chamber of a scanning electron microscope.

THE DRAWINGS The invention will now be further described with reference to the accompanying drawings, in which:

FIG. I shows the evacuation system of a typical scanning electron microscope;

FIGS. 2 and 3 illustrate how the specimen is frozen according to this invention;

FIG. 4 shows an embodiment for observing a frost free cross-section of the frozen specimen;

FIGS. 5 and 6 show embodiments for removing the frost from the surface of the frozen specimen; and,

FIG. 7 shows an embodiment for preventing the formation of frost during specimen exchange.

DESCRIPTION OF THE PREFERRED EMBODIMENTS Referring to FIG. I, an electron optical column I includes an electron gun chamber la and specimen chamber lb. Column 1 is roughly evacuated by rotary pump 2 through valve 3 and completely evacuated by diffusion pump 4 and rotary pump 5 through valve 6. A pre-evacuation chamber 7 is partitioned from the specimen chamber in column 1 by a door 8 and from the atmosphere by a cover 9. The chamber 7 is evacuated by rotary pump 2 through valve 10 and is leaked through valve 11. The following embodiment of the invention relates to the pre-evacuation chamber.

Referring to FIG. 2, a biological specimen 12 is fixed onto a specimen holder 13, into which a holder exchanging rod 14 is screwed. Thus assembled, the holder is plunged into liquid nitrogen 15 or liquid Freon in the vessel 16. The specimen is then enclosed by means of a cover 17 (shown in FIG. 3) while still submerged in the refrigerant, after which, the sealed specimen is transferred into the pre-evacuation chamber 7. Next, as

shown in FIG. 3, the cover 17 is removed under vacuum by means of exchanging rod 14, which is mounted with ball 18 and socket 19, so as to force the cover off the holder by pushing it up against projection arm 20 forming part of the pre-evacuation chamber 7. Removal of the cover is facilitated by the provision of a glass viewing window 21 forming part of the cover 9. When this is completed, the specimen is transferred to the specimen stage in the specimen chamber in column 1, the stage being maintained in the cold state, the scanning electron beam is applied, and the specimen image is observed.

Referring to FIG. 4, an interim stage 22 is maintained in the cold state by means of a liquid nitrogen tank 23 and a heat conducting rod 24. An optical microscope 25 for observing the specimen mounted on the interim specimen stage 22 is mounted on the walls of the preevacuation chamber. A manipulating rod 26, supported by a ball 27 and socket 28 for ease of maneuverability is mounted in the chamber wall. A knife 29 or needle is attached to the tip of the rod in order to cut or slice the frozen specimen. Since the knife 29 or needle must be maintained in the cold state during specimen preparation, it is necessary to either bring the knife or needle into direct contact with the stage 22 prior to carrying out the cutting or slicing operation or to connect it to the heat conducting rod 24 by means of heat conducting wire belt 30. In either case, this embodiment enables the frozen specimen to be cut or sliced as desired without frost forming on the newly exposed surface, thereby making it possible to observe the inner structure of the specimen in an almost natural state.

Referring to FIG. 5, there is shown an embodiment comprising a heating coil 31 provided in the vicinity of the interim stage upon which the specimen holder complete with specimen is mounted. Heating current is supplied to the heating coil 30 from a supply source 32 via insulators 33. By utilizing this embodiment, it is possible to defrost a whole area of the specimen surface.

Referring to FIG. 6, there is shown an embodiment comprising an infrared ray irradiating device 34 mounted in place of the manipulating rod shown in FIG. 4. The device 34 is mounted on a roller 35, again for ease of maneuverability. Consequently, the infrared rays emitted from the lamp 36 are focused by two builtin lenses 37 and 38 and can be optionally directed on any desired part of the frozen specimen surface. In this way, it is possible to defrost the specific area for observation hardly affecting the temperature of the specimen as a whole.

The embodiment as shown in FIG. 7 is designed to prevent frost forming in the specimen chamber during specimen exchange. In the conventional apparatus, it is necessary to expose the pre-evacuation chamber to the atmsophere in order to retrieve the examined specimen and replace it with a new one. Consequently, the interim specimen stage becomes coated with frost; moreover, the sublimation of this frost, as a result of subsequent evacuation, causes frost to form on the new specimen. To prevent this from occurring, this invention employs the method whereby dry gas from a gas supply source 39 is supplied to the chamber via the leak valve 11 during the time the specimen is being exchanged.

Having thus described the invention with the detail and particularity as required by the Patent Laws, what is desired protected by Letters Patent is set forth in the following claims.

We claim:

1. An apparatus for treating a frozen biological specimen or the like in a scanning electron microscope having a specimen chamber and a specimen stage therein comprising a pre-evacuation chamber partitioned from the specimen chamber of the microscope, an interim stage disposed within said pre-evacuation chamber, a specimen holder for holding the specimen, said holder being mounted on said interim stage, means for maintaining the interim stage and the holder sufficiently cold to maintain the sample frozen, a cutting tool attached to the pre-evacuation chamber for cutting the frozen specimen, a heating means for defrosting a surface of the frozen specimen, and means for transferring the treated specimen from the interim stage to the specimen stage of the specimen chamber.

2. An apparatus according to claim 1 in which said heating means comprises a heating coil arranged around the frozen specimen mounted on the interim stage.

3. An apparatus according to claim 1 in which said heating means comprises an infrared ray irradiating device equipped with focusing means for selectively defrosting a given area of the surface of the frozen specimen mounted on the interim stage.

4. An apparatus according to claim 1 including means for keeping said cutting tool sufficiently cold so as not to unfreeze the sample surface on contact.

5. An apparatus for treating a frozen biological specimen or the like in a scanning electron microscope having a specimen chamber and a specimen stage therein comprising a pre-evacuation chamber partitioned from the specimen chamber of the microscope, an interim stage housed in said pre-evacuation chamber, a specimen holder holding the specimen, said holder being mounted on said interim stage, means for maintaining the interim stage and the holder sufficiently cold to maintain the sample frozen, a cutting tool attached to the pre-evacuation chamber for cutting the frozen specimen, a heating means for defrosting a surface of the frozen specimen, an optical microscope mounted in the wall of the pre-evacuation chamber for observing the specimen from outside the chamber, and means for transferring the treated specimen from the interim stage to the specimen stage of the specimen chamber.

6. An apparatus for treating a frozen biological specimen or the like in a scanning electron microscope having a specimen chamber and a specimen stage therein comprising a pre-evacuation chamber partitioned from the specimen chamber of the microscope, an interim stage housed in said pre-evacuation chamber, a specimenholder for holding the specimen, said holder being mounted on said interim stage, means for maintaining the interim stage and the holder sufficiently cold to maintain the sample frozen, means for treating the frozen specimen, means for transferring the treated specimen from the interim stage to the specimen stage of the specimen chamber, and a means for supplying the preevacuation chamber of the microscope with dry gas during specimen exchange. 

1. An apparatus for treating a frozen biological specimen or the like in a scanning electron microscope having a specimen chamber and a specimen stage therein comprising a pre-evacuation chamber partitioned from the specimen chamber of the microscope, an interim stage disposed within said pre-evacuation chamber, a specimen holder for holding the specimen, said holder being mounted on said interim stage, means for maintaining the interim stage and the holder sufficiently cold to maintain the sample frozen, a cutting tool attached to the pre-evacuation chamber for cutting the frozen specimen, a heating means for defrosting a surface of the frozen specimen, and means for transferring the treated specimen from the interim stage to the specimen stage of the specimen chamber.
 2. An apparatus according to claim 1 in which said heating means comprises a heating coil arranged around the frozen specimen mounted on the interim stage.
 3. An apparatus according to claim 1 in which said heating means comprises an infrared ray irradiating device equipped with focusing means for selectively defrosting a given area of the surface of the frozen specimen mounted on the interim stage.
 4. An apparatus according to claim 1 including means for keeping said cutting tool sufficiently cold so as not to unfreeze the sample surface on contact.
 5. An apparatus for treating a frozen biological specimen or the like in a scanning electron microscope having a specimen chamber and a specimen stage therein comprising a pre-evacuation chamber partitioned from the specimen chamber of the microscope, an interim stage housed in said pre-evacuation chamber, a specimen holder holding the specimen, said holder being mounted on said interim stage, means for maintaining the interim stage and the holder sufficiently cold to maintain the sample frozen, a cutting tool attached to the pre-evacuation chamber for cutting the frozen specimen, a heating means for defrosting a surface of the frozen specimen, an optical microscope mounted in the wall of the pre-evacuation chamber for observing the specimen from outside the chamber, and means for transferring the treated specimen from the interim stage to the specimen stage of the specimen chamber.
 6. An apparatus for treating a frozen biological specimen or the like in a scanning electron microscope having a specimen chamber and a specimen stage therein comprising a pre-evacuation chamber partitioned from the specimen chamber of the microscope, an interim stage housed in said pre-evacuation chamber, a specimen holder for holding the specimen, said holder being mounted on said interim stage, means for maintaining the interim stage and the holder sufficiently cold to maintain the sample frozen, means for treating the frozen specimen, means for transferring the treated specimen from the interim stage to the specimen stage of the specimen chamber, and a means for supplying the pre-evacuation chamber of the microscope with dry gas during specimen exchange. 